Use of anti-cd100 antibodies

ABSTRACT

The invention relates to the use a BD16 and/or BB18 anti-CD100 antibody or of a chimeric or humanized or human form thereof, or a fragment thereof, for the therapy or diagnosis of a central nervous system disorder, more particularly a myelin disorder or a disease that affects oligodendrocytes, such as multiple sclerosis or HTLV-1 associated myelopathy or peripheral myelinating cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/544,031, filed Jun. 14, 2006, which was a national stage filing under35 U.S.C. 371 of PCT/EP2004/001743, filed Feb. 2, 2004, whichInternational Application designated the U.S., was published by theInternational Bureau in English on Aug. 12, 2004, and claims priority toEuropean Patent Application EP 03290247.0, filed Jan. 31, 2003, each ofwhich is incorporated by reference in its entirety herein.

The present invention relates to the use of anti-CD100 antibodies forthe treatment or diagnosis of neuroinflammatory diseases.

CD100 is a human 150-kDa homodimer expressed at the surface of mosthemopoietic cells, and its gene belongs to the Ig and semaphorin genefamilies (Bougeret et al, 1992; Herold et al, 1995). Semaphorin genesencode soluble and membrane-bound proteins, most of which have beenshown to act as chemorepellents on growth cone guidance. Delaire et al.,1998, suggested that CD100 plays a role in T lymphocyte activation.Soluble CD100 was further shown to inhibit immune cell migration(Delaire et al., 2001). This human immune semaphorin CD100 (Hall et al.,1996; Bismuth et al., 2002) is released at high levels by T lymphocytesupon activation through proteolysis by a metalloproteinase (Elhabazi etal., 2001). It was further shown to be expressed in oligodendrocytesduring myelination (Moreau-Fauvarque, 2002).

The inventors hypothesized that CD100 could play a critical role inneural-immune interactions in inflammatory situation.

Neuroinflammatory diseases, including multiple sclerosis (MS), the majorinflammatory demyelinating disease of the CNS, and myelopathy associatedwith HTLV-I infection (HAM/TSP), are characterized by myelindestruction, oligodendrocyte and axonal loss in brain and spinal cord.Presence of activated T lymphocytes together with increased expressionof inflammatory cytokines and metalloproteinases, correspond well withactive lesions in the central nervous system (CNS) of patients.Infiltrating T lymphocytes are suspected to take part in cytotoxicity,inflammatory molecule synthesis, dysregulation of oligodendrocytehomeostasis and axonal damages.

The inventors investigated in vitro the effect of chronically activatedT cells expressing sCD100, and recombinant sCD100, on human pluripotentneural precursors and on rat oligodendrocytes. These cellular contactsmimic interactions occurring during neuroinflammation between activatedT lymphocytes and oligodendrocytes or pluripotent neural precursorsstill capable of generating neurons or oligodendrocytes in adult brain(Zhang et al., 1999).

The inventors have particularly analyzed cell survival andoligodendrocyte process extensions. They have shown that the specificanti-CD100 monoclonal antibodies called BD16 and BB18 were able to blockthe interaction of soluble CD100 with its receptor in activated T cells,whereby they prevented the apoptosis of oligodendrocytes in HTLV-1associated disease.

The presence of sCD100 in the cerebrospinal fluid (CSF) and CD100expressing activated T lymphocytes in post-mortem brains from HAM/TSPpatients was also explored to support the functional relevance of theexperimental results.

On that basis the inventors now propose to use the specific anti-CD100antibodies, and especially the monoclonal antibodies called BD16 andBB18, in the treatment or diagnosis of neuroinflammatory diseases.

The treatment or diagnostic methods of the invention are useful for anysubject or patient. “Treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in which the disorder is to be prevented. Thepatient is preferably a human, preferably an adult, but the methodsaccording to the present invention can also be applied to other mammalsor vertebrates.

Anti-CD100 Monoclonal Antibodies

The preferred anti-CD100 antibodies used according to the presentinvention are monoclonal antibodies called BD16 and BB18.

BD16 is a mouse monoclonal antibody that is produced by a hybridomadeposited at the European Collection of Cell Cultures (ECACC), a HealthProtection Agency Culture Collection (HPACC), Health Protection Agency,Centre for Emergency Preparedness and Response, Porton Down, Salisbury,Wiltshire SP4 0JG, United Kingdom on Jan. 7, 1992 (deposit number 92-010801), as described in the international patent application WO 93/14125.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. Access to this deposit will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and persons determined by the Commissioner to beentitled thereto upon request. Upon allowance of any claims in theapplication, the Applicants will make available to the public, pursuantto 37 C.F.R. §1.808, sample(s) of the deposit with the HPACC. Theisolation of this antibody is described in Herold et al, 1995. Thisantibody can further be purchased from Beckman-Coulter. This antibodywas tested on immune cell migration in Delaire et al., 2001.

BB18 is another mouse monoclonal antibody that is produced by ahybridoma deposited at the European Collection of Cell Cultures (ECACC),a Health Protection Agency Culture Collection (HPACC), Health ProtectionAgency, Centre for Emergency Preparedness and Response, Porton Down,Salisbury, Wiltshire SP4 0JG, United Kingdom on Jan. 7, 1992 (depositnumber 92-01 0802), as described in the international patent applicationWO 93/14125. This deposit will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicants will make available to thepublic, pursuant to 37 C.F.R. §1.808, sample(s) of the deposit with theHPACC. The isolation of this antibody is described in Bougeret et al.,1992.

Both antibodies, which are Ig A, recognize the human form of the CD100semaphorin, via conformational epitopes. Whereas BB18 inducesinternalization of CD100, BD16 promotes its release. Whereas BB18maintains CD100 in association with kinases, BD16 dissociates thesecomplexes. BD16 is co-mitogenic with CD2 and CD3, while BB18 isco-mitogenic with PMA (phorbol ester). In spite of these differentcharacteristics and properties, both the BD16 and BB18 antibodies areuseful as blocking antibodies.

Hybrid and recombinant forms of these antibodies, as well as fragmentsthereof (e.g., Fab, F(ab′)2 and Fv), are also useful according to thepresent invention, so long as they exhibit the desired biologicalactivity.

This means that the antibodies or fragments thereof useful according tothe invention are able to bind human CD100 with essentially the sameaffinity and specificity than the BD16 or BB18 antibody and, when usedfor purposes of prevention or treatment of a central nervous system orperipheral nervous system inflammatory disorder, exhibit essentially thesame blocking property with regard to the activity of human CD100 thanthe BD16 or BB18 antibody.

Anti-CD100 Chimeric or Humanized Antibodies

Especially with regard to the therapeutics in humans, it may beadvantageous to use chimeric or humanized forms of BD16 or BB18antibodies.

Hybrid and recombinant antibodies may be produced by splicing a variable(including hypervariable) domain of an anti-CD100 antibody with aconstant domain (e.g. “humanized” antibodies), or a light chain with aheavy chain, or a chain from one species with a chain from anotherspecies, or fusions with heterologous proteins, regardless of species oforigin or immunoglobulin class or subclass designation, so long as theyexhibit the desired biological activity (See, e.g., Cabilly et al., U.S.Pat. No. 4,816,567; Mage and Lamoyi, in Monoclonal Antibody ProductionTechniques and Applications, pp. 79-97 (Marcel Dekker, Inc., New York,1987)).

An aspect of the present invention encompasses using “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass.

“Humanized” forms of the BD16 or BB18 antibodies are specific chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from mouse immunoglobulin. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementarity-determining region(CDR) of the recipient are replaced by residues from a CDR of thenon-human species (donor antibody) having the desired specificity,affinity, and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody comprisessubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (see Berger et al., 2002 for a review of the availabletechniques).

Full Human Anti-CD100 Antibodies

In another aspect of the invention one may use human anti-CD100antibodies, i.e. human anti-CD100 antibodies recognizing similar orclosely related epitope(s) to BD16 or BB18. These antibodies areobtainable from a human immunoglobulin gene library.

Several methods for producing human antibodies are described e.g. in theInternational Patent Application WO 01/040306.

These methods include the phage display methods and nonhuman transgenicmice expressing genes of the human immune system. The methods typicallywork by immunizing a nonhuman transgenic animal having humanimmunoglobulin genes. The animal expresses a diverse range of humanantibodies that bind to the antigen. Nucleic acids encoding the antibodychain components of such antibodies are then cloned from the animal intoa display vector. Typically, separate populations of nucleic acidsencoding heavy and light chain sequences are cloned, and the separatepopulations then recombined on insertion into the vector, such that anygiven copy of the vector receives a random combination of a heavy andlight chains. The vector is designed to express antibody chains so thatthey can be assembled and displayed on the outersurface of a displaypackage containing the vector. For example, antibody chains can beexpressed as fusion proteins with a phage coat protein from theoutersurface of the phage. Thereafter, display packages can be screenedfor display of antibodies binding to a target.

Useful cDNA libraries for human immunoglobulin heavy chain and lightchain variable regions can also be built and expressed in a variety ofdifferent hosts, such as bacteria (e.g. E. coli) or viruses, such as apoxvirus (Smith et al, 2001) or a phage.

In the phage display methods, human immunoglobulin heavy chain and lightchain variable regions may be cloned, combinatorially reasserted,expressed and displayed as antigen-binding human Fab or scfv (“singlechain variable region”) fragments on the surface of filamentous phage,especially phage M13, Fd and F1. See Rader et al., (1997); Aujame etal., (1997); Hoogenboom, (1997); de Kruif et al., (1996); Barbas et al.,(1996); Winter et. al., (1994). The phage-displayed humanantigen-binding fragments may then be screened for their ability to binda chosen antigen.

Therapy

In a first aspect of the invention it is provided a method for theprevention or treatment of a central (CNS) or peripheral (PNS) nervoussystem inflammatory disorder, which method comprises administering aneffective amount of BD16 and/or BB18 anti-CD100 antibody or of achimeric or humanized form thereof, or a human form thereof, as well asfragments thereof to a patient in need of such treatment.

Another subject of the invention is the use of a BD16 and/or BB18anti-CD100 antibody or of a chimeric or humanized form thereof, or ahuman form thereof, as well as fragments thereof, for the preparation ofa medicament useful in the prevention or treatment of a central orperipheral nervous system inflammatory disorder.

Infiltration of the CNS or PNS by activated T lymphocytes can sustainthe onset and progression of inflammatory diseases. The inventors hereinpoint out the primary role of immune semaphorin in demyelination assoluble CD100, produced by viraly-activated T lymphocytes, induced theapoptotic death of human neural precursors (with oligodendrocytedifferentiating ability) and the progressive decrease in processextensions followed by death of immature oligodendrocytes. The specificexpression of sCD100 in CSF of HAM/TSP patients and the presence ofnumerous infiltrating CD100/CD45RO-positive T lymphocytes in theirspinal cord (in contrast with other neurological cases) substantiate thepotential deleterious effect of CD100 in neuroinflammatory demyelinatingdiseases. The anti-CD100 antibodies may further be useful to preventoligodendrocyte impairment or impairment of other peripheral myelinatingcells, therefore extension of neurological disorders.

Accordingly the CNS or PNS inflammatory disorder the present inventionis aimed at is preferably a myelin disorder or a disease that affectsoligodendrocytes or other myelinating cells, e.g. peripheral myelinatingcells. More particularly prevention or treatment of multiple sclerosisor HTLV-1 associated myelopathy is encompassed.

One can also cite more generally neuroinflammatory disorders, as well asoligodendrogliomas and leucodystrophies, Guillain-Barrésyndrome,Alexander disease, Canavan disease, Krabbe disease, Pelizaeus-Merzbacherdisease, Zellweger disease, Refsum disease, CACH disease, X-linkedadrenoleucodystrophy, adrenoleucodystrophy, adrenomyeloneuropathy orleucodystrophies of undetermined origin, or polyradiculoneuritis as wellas chronic polyradiculoneuritis.

In another aspect of the invention, the disorder is a post-trauma myelindisorder, as well as CNS or PNS lesions, for example caused by spinalcord injury or stroke.

Another subject of the invention is thus a method for stimulating axonalmyelination, which method comprises administering to a patient in needof such treatment a therapeutically active amount of a BD16 and/or BB18anti-CD100 antibody.

Pharmaceutical Compositions

Anti-CD100 antibodies can be formulated in pharmaceutical compositions,for a topical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected,optionally directly into the central nervous system or in the brain,e.g. by intracerebroventricular injection for dispersion into otherareas.

The suitable pharmaceutical compositions may be in particular isotonic,sterile, saline solutions (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride and the like or mixtures ofsuch salts), or dry, especially freeze-dried compositions which uponaddition, depending on the case, of sterilized water or physiologicalsaline, permit the constitution of injectable solutions.

The doses of anti-CD100 antibodies used for the administration can beadapted as a function of various parameters, and in particular as afunction of the mode of administration used, of the relevant pathology,or alternatively of the desired duration of treatment.

To prepare pharmaceutical compositions for antibody therapy, aneffective amount of the protein may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

Examples of pharmaceutical formulations are provided hereafter.

Pharmaceutical compositions comprise an effective amount of ananti-CD100 antibody in a pharmaceutically acceptable carrier or aqueousmedium.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

As used herein, a “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, intrathecal, intracerebroventricular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

The anti-CD100 antibody may be formulated within a therapeutic mixtureto comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including creams.

Other routes of administration are contemplated, including nasalsolutions or sprays, aerosols or inhalants, or vaginal or rectalsuppositories and pessaries.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of the antibodies. The formation anduse of liposomes is generally known to those of skill in the art, and isalso described below.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

The following information may also be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs as a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynon-specific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membrane, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Diagnosis

The inventors demonstrated the presence of sCD100 in the cerebrospinalfluid (CSF) and CD100-expressing activated T lymphocytes in post-mortembrains from HAM/TSP patients.

On that basis the present invention provides an in vitro method fordiagnosis or determination of the evolution of a central nervous system(CNS) or peripheral nervous system (PNS) inflammatory disorder, whichmethod comprises assaying a CD100 semaphorin in a biological sample of atest subject, compared with the level of a CD100 semaphorin in abiological sample of a control subject, wherein an increased level ofCD100 is indicative of said disorder or of a poor prognosis, wherein theassay is an immunoassay employing a BD16 and/or BB18 anti-CD100antibody, a chimeric or humanized form thereof, or a fragment thereof.

A “biological sample” is a fluid from a subject, including serum,plasma, blood, spinal fluid, cerebrospinal fluid, urine, ascites,pleural effusion, amniotic fluid, tears or saliva, or a tissue extractor a tissue or organ biopsy such as brain, spinal cord or nerves.

“A subject” or “a patient” is a vertebrate, e.g. a mammal, preferably ahuman being, regardless of his/her age, sex and general condition.Children and infants are also encompassed. The test subject may beasymptomatic, may be considered likely to develop the disease orcondition. Subjects with a suspicion of myelin disorder or subjects whohave already shown symptoms of the disease or condition can also betested. Subjects who are predisposed to developing a CNS or PNSinflammatory disorder naturally are a preferred target.

The <<control subject>> may be a healthy subject or a subject withoutany myelin disorder. In order to determine the evolution of a CNS or PNSinflammatory disorder, it may be very useful to test a subject for theexpression of CD100 or of a receptor thereof, and to monitor the effectof a drug or the spreading of the condition, by testing him/her a secondtime, e.g. a few weeks later. In that case the results of the secondtest are compared with the results of the first test, and in generalalso with the results obtained with a “healthy” subject. The “controlsubject” then refers either to the same test subject or to a “healthysubject”.

“A CNS or PNS inflammatory disorder” includes any neuroinflammatorydisorder, myelin disorder, or disease that affects oligodendrocytes, orother myelinating peripheral cells as described above. Diagnosis ormonitoring of the evolution of multiple sclerosis or HTLV-1 associatedmyelopathy is more particularly contemplated. The in vitro methodprovided herein is particularly advantageous for that purpose, in thatit makes it possible to easily and quickly assay the CD100 in a fluidsample, such as a CSF sample for instance.

In a preferred embodiment the invention provides an in vitro method fordiagnosis or monitoring a HTLV-1 associated myelopathy in a patient,wherein the CD100 semaphorin is assayed in a CSF sample of the patientby means of an ELISA technique employing a BD16 and/or BB18 anti-CD100antibody, a chimeric or humanized form thereof, or a human form thereof,as well as fragments thereof.

The term “diagnosis” refers to the determination or the confirmation ofa disease or condition in a subject. The term “poor prognosis” meansthat the condition has worsened.

Methods for immunoassaying the CD100 semaphorin protein or a receptorthereof are well known by one skilled in the art. Such assays includecompetition, direct reaction, or sandwich type assays. Varioustechniques including Western blots, enzyme-labeled and mediatedimmunoassays, such as ELISAs; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, etc., may be employed. ELISAs are preferred. Thereactions generally include revealing labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound CD100in a liquid phase from a solid phase support to which antigen-antibodycomplexes are bound. Solid supports which can be used in the practice ofthe invention include substrates such as nitrocellulose (e.g., inmembrane or microtiter well form); polyvinylchloride (e.g., sheets ormicrotiter wells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like.

Typically, a solid support is first reacted with a solid phase component(i.e. an anti-CD100 antibody) under suitable binding conditions suchthat the component is sufficiently immobilized to the support accordingto methods well known to those skilled in the art. After reacting thesolid support with the solid phase component, any non-immobilizedsolid-phase components are removed from the support by washing, and thesupport-bound component is then contacted with a biological samplesuspected of containing ligand moieties (i.e. CD100 molecules toward theimmobilized antibodies) under suitable binding conditions. After washingto remove any non-bound ligand, a secondary binder moiety is added undersuitable binding conditions, wherein the secondary binder is capable ofassociating selectively with the bound ligand. The presence of thesecondary binder can then be detected using techniques well known in theart.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with an anti-CD100 antibody. A biologicalsample containing or suspected of containing CD100 molecules is thenadded to the coated wells. After a period of incubation sufficient toallow the formation of antibody-antigen complexes, the plate(s) can bewashed to remove unbound moieties and a detectably labeled secondarybinding molecule added. The secondary binding molecule is allowed toreact with any captured sample CD100 molecules, the plate washed and thepresence of the secondary binding molecule detected using methods wellknown in the art.

Thus, in one particular embodiment, the presence of bound CD100molecules from a biological sample can be readily detected using asecondary binder comprising another antibody, that can be readilyconjugated to a detectable enzyme label, such as horseradish peroxidase,alkaline phosphatase or urease, using methods known to those of skill inthe art. An appropriate enzyme substrate is then used to generate adetectable signal. In other related embodiments, competitive-type ELISAtechniques can be practiced using methods known to those skilled in theart.

Assays can also be conducted in solution, such that the antigenicpolypeptides and antibodies specific for those proteins form complexesunder precipitating conditions. In one particular embodiment, antibodiescan be attached to a solid phase particle (e.g., an agarose bead or thelike) using coupling techniques known in the art, such as by directchemical or indirect coupling. The antibody-coated particle is thencontacted under suitable binding conditions with a biological samplesuspected of containing CD100 molecules. The particle-antigen-antibodycomplexes aggregate and can be precipitated and separated from thesample using washing and/or centrifugation. The reaction mixture can beanalyzed to determine the presence or absence of antibody-antigencomplexes using any of a number of standard methods, such as thoseimmunodiagnostic methods described above.

The above-described assay reagents, including the anti-CD100 antibodies,can be provided in kits, with suitable instructions and other necessaryreagents, in order to conduct immunoassays as described above. The kitcan also contain, depending on the particular immunoassay used, suitablelabels and other packaged reagents and materials (i.e. wash buffers andthe like). Standard immunoassays, such as those described above, can beconducted using these kits.

The figures and examples illustrate the invention without restrictingits scope.

LEGENDS OF FIGURES

FIG. 1: CD100-induced death in human neural precursors. This figureshows apoptotic death in human neural precursors following transientcontact with CD100-producing T lymphocytes (TUNEL positive cells) ortreatment with soluble CD100 recombinant protein (active caspase-3positive cells) (48 h post-treatment). Count (AnalySIS®) of total (nonhatched bar) and active caspase-3 positive cells (hatched bar) isrepresented (mean±SEM, representative experiment).

FIG. 2: CD100-induced alteration of oligodendrocyte process. This figureshows progressive loss of process extensions in immature ratoligodendrocytes (OL) following transient contact with CD100-producing Tlymphocytes or treatment with soluble CD100 recombinant protein (24 hand 48 h post-treatment). The measure of OL process is representedfollowing each treatment (mean±SEM, 10-20 measures, 3 experiments).

FIG. 3: CD100-induced death of immature-OL. This figure shows thattransient contact with CD100-producing T lymphocytes or treatment withsoluble CD100 recombinant induced loss of OL (48h post-treatment). Thetotal OL loss following each treatment is represented (mean±SEM OL perfield, representative experiment).

FIG. 4: Involvement of CD100 in T cell-induced death in neuralprecursors and oligodendrocytes. This figure shows reduction of Tcell-induced death in neural precursors (FIG. 4A) and oligodencrocyte bytreatment with CD100-blocking antibody BD16. FIGS. 4B and 4C show thattreatment with sCD100 recombinant protein mimicked the T cell-induceddeath of neural precursors (B) and oligodendrocytes (C) in adose-dependent manner.

FIG. 5: Involvement of plexin-B in T cell-induced death of neuralprecursors. A-Reduction of neural cell death by anti-plexin-B1 antibody;no effect of anti-neuropilinl antibody (count of active caspase-3positive cells: mean±SEM per field). B-Detection, by RT-PCR, of Sema3AmRNA in neural cells but not in T cells. C-Detection by RT-PCR andrestriction enzyme digestion of plexin B family mRNA in human neuralcells: Pstl and Sau-3AI generated 171 pb and 129 bp, or 205 bp and 95 bpproducts, respectively from the 300 bp product common to Plexin-B1,plexin-B2 and plexin-B3 gene .D-Plexin-B1 immunodetected (FACS analysis)on human neural precursors.

FIG. 6: Levels of sCD100 IU/ml in normal subjects or patients withneuroinflammatory diseases (MS=multiple sclerosis;PRN=polyradiculoneuritis).

EXAMPLES Example 1 Blockade of Neural Progenitor and OligodendrocyteApoptosis by BD16 Anti-CD100 Antibody in HTLV-1 Associated MyelopathyMethods

To mimic a crosstalk between neural and immune cells, the inventors usedan experimental paradigm consisting of transient cocultures ofchronically activated T lymphocytes (HTLV-1 infected, no virusproductive) with human multipotent neural precursors or rat primaryoligodendrocytes. Such cell contact mimics interaction occurring in thebrain between activated T cells and oligodendrocytes or multipotentneural progenitors capable of differentiating, in adult, intooligodendrocytes. The physiological relevance of the experimental datawas examined by analyzing post mortem spinal cord and CSF from patientssuffering neuroinflammatory disease, myelopathy associated with HTLV-1myelopathy (HAM/TSP).

Model of T-neural cell interaction: The human pluripotent neuralprecursor cell line Dev, has the ability to differentiate into neurons,astrocytes and oligodendrocytes (Derrington et al., 1998; Buzanska etal., 2001). Primary culture of rat glial cells (Szymocha et al., 2000)contained 35-54% oligodendrocytes, the remaining cells corresponding toglial acidic fibrillary protein (GFAP)-positive astrocytes. Theoligodendrocytes (OL) comprised the distinct phenotypic stages ofoligodendrocytes identified by a panel of cell specific antibodies(immunochemistry or flow cytometry, see below): pre-OL (OL precursor)expressing NG2 (10-15%), immature-OL expressing galactocerebroside(GalCer, 17-25%) and cyclic nucleotic phosphodiesterase (CNPase),mature-OL (premyelinating OL) expressing myelin associated glycoprotein(MAG, 3-15%) and myelin basic protein (MBP).

These neural cells were transiently co-cultured (Giraudon et al., 2000)with: 1-the human CD4+CD25+ no virus-productive T-cell line C8166/45activated by HTLV-I infection (Popovic et al., 1983) which releasedsoluble CD100 (sCD100) detected by ELISA (Hall et al., 1996) (1400ng/ml); 2-the non activated CD4+ T cell line CEM. The T cells wereeliminated by washing of neural culture and their elimination verifiedby the absence of CD4 antigen (flow cytometry). The neural cells werealso treated with recombinant soluble CD100 (sCD100r) (0.6-10 ng/μlconcentration) from transfected Jurkat cells. (producing 800 ng/ml),used as such or purified on CD100 BB18-mAb column. Experiments wererepeated three times.

Patients: The CNS tissue samples from 3 HAM/TSP patients and 2non-infected patients (Parkinson disease, car crash) were examined forCD100-expressing T cells. Paraffin-embedded spinal cords were examinedusing routine (hematoxylin-eosinsafran) and Luxol Fast Blue for myelindetection. Spinal cord from these patients displayed moderate to markedatrophy in the thoracic level, thickening of the leptomeninges,perivascular infiltrates of immune cells, as previously described inCartier et al., 1997. Degeneration of the lateral cortico-spinal andspinocerebellar tracts, diffuse loss of myelin and axons andastrocytosis were observed in the three cases.

Characteristics of HAM/TSP Patients:

CNS neuropathology atrophy inflam. Age/ Duration myelin/axonal sex ofillness Cause of death loss case 1 62/F 15 years Pulmonary embolism ++++++ +++ case 2 35/H  4 years Mesenteric thrombosis ++ +++ ++ case 3 65/F 8 years Pneumonia + + +

The CSF from patients suffering from TSP/HAM (9), meningitis (4) orencephalomyelitis (3) were examined for the presence of sCD100 by asandwich ELISA as previously described in Delaire et al, 2001. AllHAM/TSP patients tested were positive for HTLV-1 provirus.

Immunodetection: Astrocytes and oligodendrocytes were identified byimmunofluorescence, in flow cytometry or on culture slide (Bougeret etal, 1992; Herold et al, 1995), with monoclonal antibodies (anti-CNPase,Sigma; anti-GalCer, anti-NG2, Chemicon Int.; anti-MAG, BoehringerMannheim; anti-MBP, Serotec) or rabbit polyclonal antibody (anti-GFAP,Dako). Semaphorin receptors were detected with monoclonal anti-humanplexin-B1 (N18-Tebu) and anti-MAM neuropilin-1 rabbit polyclonalantibody (Bagnard et al., 2001). T lymphocytes were detected in spinalcord sections from HAM/TSP patients by immunofluorescence withanti-CD45RO monoclonal antibody (clone UCHL1, Dako), and an anti-CD100polyclonal antiserum, directed against the intracellular portion of theprotein (AA799-813). After dewaxing with toluene and ethanol, sectionswere incubated in blocking solution (1% BSA, 0.3% triton, 1 h), thenwith specific antibodies (4° C./over night). Anti-mouse Alexa546 oranti-rabbit Alexa488 labeled antibodies (Molecular Probes) were thenapplied.RNA detection: mRNA were detected by extraction followed by reversetranscription and polymerase-chain reaction (RT-PCR) and Southernblotting using appropriate [³³P] dATP-5′ end-labeled internaloligonucleotide probes, as previously described in Szymocha et al.,2000. Oligonucleotide primers were chosen from their mRNA sequences(GenBank access numbers HSU60800 for CD100, NM002663 for plexinB1,NM012401 for plexin-B2, AF149019 for plexin-B3, L26081 for Sema-3A,NM01101 for β-actin, used as control). One set of oligomer was selectedfor amplification of a common 300 bp amplicon in the three plexin-BmRNA.Apoptosis analysis: In each experiment, apoptotic death was detected bythe TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-endlabeling)-method performed on culture slide (Promega), immunodetectionof the executer caspase, active-caspase-3 (rabbit serum—Pharmingen) anddetection of apoptotic bodies with Dapi (4′, 6-diamidino-2 phenylindol,Sigma, 1 μg/ml) staining of nucleus. Co-detection of TUNEL,active-caspase-3 and OL-markers was also performed.Statistical analysis: Total cells (Dapi staining), TUNEL andactive-caspase-3-positive cells were counted in neural precusors andprimary glial cells (15-20 microscope fields, 800-1000 cells, 3experiments) using AnalySiS 3.2 software. Measure of oligodendrocyteprocess number and length was performed by using the same software. Thevalues were expressed as mean±SEM. Difference between groups werecalculated with the Student's t-test.

Results

The effect of soluble CD100 was investigated on human pluripotent neuralprecursors and rat primary oligodendrocytes by analyzing neural cellmorphology and survival after contact with the chronically activated Tcells producing high level of sCD100, C8166/45, or the non-activated andnon-CD100-producing T cells, CEM. Apoptotic death of the human neuralprecursors was induced by C8166/45 T cells, as evidenced at 48 h post Tcell-contact by the presence of apoptotic bodies in TUNEL positive cellsand by detection, using immunofluorescence, of an enhanced number ofactive-caspase-3 positive cells (25.5±8.8 positive cells per fieldversus 11.3±3.7 in untreated cells) in three independent experiments(FIG. 1, a representative experiment). This corroborated the decrease inthe total cell number (24±4.4% neural cell loss at 48 h post T cellcontact and 58.5±9.2% at 72 h). CEM T cells had no effect on theseneural precursors.

CD100-producing T cells also induced damages in rat primary glialculture. A progressive collapse and loss of process extensions wereobserved in GalCer-positive cells (immature-OL). The length and numberof process extensions of these immature-OL were progressively reduced 24h and 48 h post-contact (FIG. 2), as revealed in three independentexperiments by the relative measure of their length (1.1±0.1 μm versus2.7±0.2 μm in untreated glial culture at 48 h) and the count ofprocesses (1.3+0.2 process per cell versus 4.3±0.5 in untreated glialculture at 48 h). In addition, an apoptotic death was observed at 48 hby the detection of TUNEL-positive cells and counting theactive-caspase-3 positive cells (19.7±8.1 per field versus 7.3±5.5 inuntreated cultures, 3 experiments). This resulted in the loss of a partof oligodendrocyte population, as shown by the reduced number ofoligodendrocytes per field (FIG. 3-, a representative experiment) andthe reduction in the percentage of total oligodendrocytes in the glialculture (26.8±3.6% loss at 48 h, 3 experiments). Interestingly, pre-OL(NG2-positive) and mature-OL (MBP-positive, FIG. 3-B) were not sensitiveto T-cell induced apoptotic death in contrast with immature-OL(GalCer-positive). The number and morphology of astrocytes wereunchanged. The CEM T cells had no effect on the neural precursors andglial culture.

Several lines of evidence indicated that sCD100 was involved in these Tcell-induced damages. The anti-CD100 BD16 mAb was able to antagonize theT cell-induced effects on neural precursors and immature-OL when addedin co-cultures. In fact, BD16-antibody dramatically decreased, in adose-dependent manner, the number of apoptotic neural precursors at 48 hpost-contact with T cells (FIG. 4-A) and reduced the cell loss(41.5±0.7% at 72 h versus 62±7.1% in antibody-untreated co-culture).Similarly, treatment of the rat co-culture with BD16-antibody reducedthe number of TUNEL-positive cells and rescued immature-OL(CNPase-positive-cells). This resulted in a decrease in the Tcell-induced oligodendrocyte loss (15.5±4.9% versus 26.3±5.4% inantibody-untreated co-culture).

The direct effect of CD100 was demonstrated by treating the neural cellswith recombinant sCD100 (sCD100r, 0.6-10 ng/μl, three experiments),released as a dimer from transfected Jurkat cells (Delaire et al.,2001). Similarly to contact with CD100-producing T cells, treatment withsCD100r induced apoptosis of neural precursors as demonstrated by theincreased number of active-caspase-3 positive cells (25.3±3.6 per fieldin culture treated with 4.2 ng/μl sCD100r versus 11.1±2.3 in untreatedculture) (FIG. 1—a representative experiment) and loss of theseprecursors (43.5±13.4% cell loss on 72 h). sCD100r induced apoptosis ofneural precursors in a dose dependent manner as shown in FIG. 4-B.Treatment of glial primary culture with sCD100r (three experiments) alsoresulted in oligodendrocyte damage and apoptosis (FIGS. 2, 3, 4). Adecrease in process number (1.4±0.2 process per oligodendrocyte at 48 hversus 5.3±0.4 in untreated culture) and their relative length (1±0.2 μmat 48 h versus 2.1±0.3μm in untreated culture) were detected inimmature-OL (GalCer-positive, FIG. 2). This was followed by an increasednumber of oligodendrocytes expressing active-caspase-3 (15±2.3 positivecells per field in culture treated with 4.2 ng/μl sCD100r versus 6±1.9positive cells in untreated culture). In fact, sCD100r reduced thenumber of oligodendrocytes per field, as shown in FIG. 3 (onerepresentative experiment), and the number of total oligodendrocytes(29±4% loss at 48 h post-treatment). Higher dose of sCD100r (10 pg/μl)induced the death of 37% total oligodendrocytes and 64% neuralprecursors at 72 h. Double-labeling (TUNEL and OL-markers) indicatedthat the immature-OL were sensitive to sCD100r similarly toCD100-producing T cells. The inventors also observed that SCD100rinduced apoptosis of oligodendrocytes in a dose dependent manner (FIG.4-C). In contrast, no detectable change was observed in astrocytes. Thetreatment of these human and rat neural cells with a supernatant fromJurkat permanently transfected with an unrelated molecule, CD27(sControl), did not induce any change.

CD100 could mediate its effect through receptors of neuropilin andplexin families present on immune and neural cells. Neuropilins act asco-receptors with plexins, while plexins alone behave as fullyfunctional signal transducers for both transmembrane and secreted formsof semaphorins (Tamagnone et al., 1999). As neuropilin-1 is present onhuman neural precursors and mediates Sema 3A-induced apoptosis of thesecells (Bagnard et al., 2001), neuropilin-1 and Sema 3A could besuspected in the T cell-induced apoptosis of neural precursors. However,when added in the T-neural cells co-culture, anti-MAM neuropilin-1antibody (anti-NPL-1), shown to block the Sema 3A-induced neuralprecursors death (Bagnard et al., 2001), had no significant effect onthe rate of death induced by CD100-producing T cells (FIG. 5-A). Inaddition, the possibility of an effect of anti-CD100 antibody BD16 onSema 3A activity (Delaire et al., 2001), was ruled out by the absence ofSema 3A expression in T cells (RT-PCR, FIG. 5-B). These observationsexcluded the implication of neuropilin-1/Sema 3A in the T cell-mediateddeath. Involvement of plexins, in particular plexin-B1 identified as areceptor for sCD100 (Derrington et al., 1998), was further investigated.Presence of mRNA coding plexin-B1, B2 or B3 in human neural precursorsand human fetal cortex (used as positive control) was checked by RT-PCR.Taking into account the high homology of RNA sequence between thesethree plexins, oligomers were selected for their ability to amplify acommon 300 bp amplicon in neural precursors and human fetal brain.Amplicons were further identified by restriction enzymes Pstl andSau-3AI which can generate 171 bp and 129 bp products in the plexin-B1and plexin-B3 amplicons, and 205 bp and 95 bp products in the plexin-B1and plexin-B2 amplicons, respectively. As shown in FIG. 5-C, Pstlgenerated the expected products from 300 bp amplicon, demonstrating thepresence, in human neural precursors, of plexin-Bi or -B3 mRNA and -B2at a less extent, as shown in human fetal cortex. The presence ofplexin-B1 at the cell membrane was confirmed by immunodetection on aliveneural precursors and flow cytometry on 50-54% cells (FIG. 5-D). Theanti-plexin-B1 antibody-treatment of neural precursors, throughout theco-culture, significantly reduced the number of apoptotic neural cells(FIG. 5-A) and cell loss (5% versus 25.4% in untreated co-culture, at 48h), indicating its possible involvement in this CD100-producing T cellmediated-apoptosis. The absence of anti-plexin-B1 effect on T-mediatedoligodendrocyte apoptosis (despite the expression of plexin-B1) could bedue to the uncapacity of anti-human plexin-B1 to block rat plexinB1 orto the involvement of another plexin receptor.

Thus, these observations demonstrated that sCD100 produced by activatedT lymphocytes severely impair immature-OL and human neural precursors,identifying semaphorin as a molecule of T cell-neural cell crosstalkduring inflammation. The functional relevance of these experimental datawas investigated in patients suffering neuroinflammatory diseasecompared to patients with other neurological disease.

The inventors particularly studied HAM/TSP patients as sCD100 is highlyreleased by T lymphocytes chronically infected with HTLV-1. Expressionof sCD100, analyzed in the cerebrospinal fluid (CSF) from patients withHAM/TSP (n=9), meningitis (n=4) or encephalomyelitis (n=3) by ELISA,revealed the presence of sCD100 at a level of 97.7±23.1 μg/ml inHAM/TSP' CSF (Table 1).

TABLE 1 Soluble CD100 in the CSF from HAM/TSP patients detected by ELISA(Hall et al., 1996). Patients with other neurological diseases werenegative for CD100 detection in CSF HAM/TSP patient n^(o) CD100 (ng/ml)1 132.6 ± 29.9  2 66.6 ± 14.2 3 158.3 ± 17.2  4 79.6 ± 36.3 5 123.9 ±42.7  6  60 ± 9.9 7 62.9 ± 14.2 8 57.9 ± 16.3 9   75 ± 27.6

This was in sharp contrast with meningitis and encephalomyelitis' CSFwhere CD100 was undetectable. Interestingly, these sCD100 levels arecomparable to that found in T cells activated with CD3 crosslinking (40ng/ml). The T cells present in CSF of HAM/TSP patients which arechronically activated by the viral protein Tax (Moritoyo et al., 1999),and produce high level of MMP could be one source of sCD100 expressiondetectable in those patients and not in patient with nonneuroinflammatory disease. In addition, CD100 positive infiltratingcells were detected in post-mortem CNS from three HAM/TSP patients. Inthese patients, the level of immune cell infiltration correlates theextent of myelin/axonal loss and spinal cord atrophy (see Materialssection). Analysis of immune cells infiltrating the spinal cord of thesepatients, using anti-CD100 and anti-CD45RO, a marker of activated Tcells, showed the co-expression of these molecules. The highest densityof double-labeled T lymphocytes was found in meningia and around bloodvessels. Double-labeled T lymphocytes were also observed in parenchymaboth in grey and white matters. Interestingly, astrocytosis in the whitematter and around blood vessels was revealed by GFAP immunostaining, andthe inventors had detected the metalloproteinase MMP-9 withinastrocytosis in one of these HAM/TSP patients in a previous work²². Incontrast, there is few CD45RO-positive but all CD100-negative Tlymphocytes in spinal cords from uninfected patients.

These results show that T lymphocytes producing sCD100 induced apoptosisof neural progenitors and immature oligodendrocytes after a progressivedecrease of their process extensions. Blockade by specific antibody ofsCD100 from activated T cells, demonstrated that this immune semaphorinhas the ability to trigger oligodendrocyte process alteration, andneural cell apoptosis likely through receptor of the plexin family. Thepresence of sCD100 in the CSF and of CD100 expressing T lymphocytesinfiltrating the spinal cord of HAM/TSP patients points out thepotential deleterious effect of sCD100 in the CNS during inflammation.

Example 2 BB18 Induces internalization of CD100

Because membrane CD100 (mCD100) is down regulated from the surface ofresting T-cells by the addition of CD100 mAb BD16 or BB18 the inventorsstudied the fate of CD100 following its surface labeling with biotin.

Freshly isolated human PBMC were surface-labeled with biotin. Cells werethen incubated with the indicated ascite mAb (1/100 final dilution) orwith PMA (1 ng/ml). After 1 hour of incubation, cell supernatants werecollected and supplemented with protease inhibitors, subjected tofiltration to eliminate cell debris followed by immunoprecipitation witha mixture of BD16 and BB18 mAb. In parallel, cells were washed twicewith ice-cold PBS and lysed in NP40-based lysis buffer plus proteaseinhibitors before immunoprecipitation with the BD16/BB18 mixture. TheCD100 ipp were subjected to SDS-PAGE and western blot using theperoxidaseconjugated streptavidin detection method. Similar results wereobtained with PBMC isolated from most of the individuals tested.

The inventors thereby detected a soluble form of CD100 (sCD100) from theculture supernatants of cells incubated with BD16, or with the phorbolester PMA whereas the incubation of resting T-cells with BB18 ratherprovoked the internalization of the molecule.

Example 3 Detection of sCD100 in Neuroinflammatory Diseases

The inventors further tested 45 serum and 22 plasma (harvested on EDTA)from normal individuals were assayed (ELISA) for the presence of solubleCD100. Whereas the level of sCD100 in plasma was undetectable in most ofthem (18/22), the inventors were able to measure the level of sCD100 inthe serum with a mean of 121 nanogramme/ml and standard deviation of 26ng/ml. This was performed in comparison with a standard sCD100 afterpurification of sCD100 from a transfected cell line, producing highlevels od sCD100 that was purified on BB18 mAb affinity column.

The inventors next used a test with a different standard sCD100(freeze-dried) giving the value of CD100 in International Unit (IU) perml. Thus they could measure the levels of sCD100 in the sera of multiplesclerosis (MS) patients. The level of sCD100 in MS serum (21.66 IU/ml,SD 4.52) was at least twice the level observed in normal individuals(5.60 IU/ml, SD 3.55). In addition, the inventors tested the plasma of 5patients with polyradiculoneuritis (PRN) in crisis. The level of sCD100was even higher (33.84 IU/ml, SD22.88) whereas the level of sCD100 innormals was unsignificant (1.91 IU/ml, SD 1.38). The results of theseexperiments are shown on FIG. 6.

These data confirm that sCD100 has a deleterious effect on the nervoussystem, particularly in neuro-inflammatory diseases involvingmyelinating cells such as HAM (HTLV1 associated myelopathy), MS and PRN.

These data further support the use of neutralizing anti-CD100 antibodiessuch as BD16 or BB18 monoclonal antibodies, to prevent or inhibit sCD100effect.

The above ELISA method provides an easy way to monitor the evolution andcrisis occurring MS or HAM for instance.

REFERENCES

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1. A method for treating a neuroinflammatory disorder in a subject, saidmethod comprising administering an effective amount of an anti-CD100antibody or an antigen-binding fragment thereof to said subject, whereinsaid anti-CD100 antibody or an antigen-binding fragment thereof blocksthe interaction of soluble CD100 with plexin B1 receptor.
 2. The methodof claim 1, wherein said neuroinflammatory disorder is a central nervoussystem (CNS) inflammatory disorder.
 3. The method of claim 1, whereinsaid neuroinflammatory disorder is a peripheral nervous system (PNS)inflammatory disorder.
 4. The method of claim 1, wherein said subjecthas a high level of soluble CD100 in a biological sample taken from saidsubject when compared to a control subject.
 5. The method of claim 4,wherein said biological sample is cerebral spinal fluid (CSF) or blood.6. The method of claim 1, wherein said neuroinflammatory disorder is amyelin disorder or a disease that affects oligodendrocytes
 7. The methodof claim 1, wherein said neuroinflammatory disorder is a disease thataffects myelinating peripheral cells.
 8. The method of claim 1, whereinsaid neuroinflammatory disorder is selected from the group consisting ofan oligodendroglioma, leucodystrophy, Guillain-Barrésyndrome, Alexanderdisease, Canavan disease, Krabbe disease, Pelizaeus-Merzbacher disease,Zellweger disease, Refsum disease, CACH disease, X-linkedadrenoleucodystrophy, adrenoleucodystrophy, adrenomyeloneuropathy,leucodystrophies of undetermined origin, HTLV-1 associated myelopathy,multiple sclerosis, and polyradiculoneuritis.
 9. The method of claim 8,wherein said neuroinflammatory disorder is HTLV-1 associated myelopathy.10. The method of claim 8, wherein said neuroinflammatory disorder ispolyradiculoneuritis.
 11. The method of claim 8, wherein saidneuroinflammatory disorder is multiple sclerosis.
 12. The method ofclaim 1, wherein said neuroinflammatory disorder is a post-trauma myelindisorder of the central or peripheral nervous system.
 13. The method ofclaim 1, wherein said anti-CD100 antibody is a chimeric, humanized, orhuman antibody.
 14. A method for protecting neural progenitor cells oroligodendrocytes from cell death, said method comprising administeringto a subject an effective amount of an anti-CD100 antibody or anantigen-binding fragment thereof, and wherein said anti-CD100 antibodyor an antigen-binding fragment thereof blocks the interaction of solubleCD100 with plexin B1 receptor.
 15. The method of claim 14, wherein saidsubject has a high level of soluble CD100 in a biological sample takenfrom said subject in comparison to a control subject.
 16. The method ofclaim 15, wherein said biological sample is cerebral spinal fluid (CSF)or blood.
 17. The method of claim 14, wherein said anti-CD100 antibodyis a chimeric, humanized, or human antibody.